This invention is in the field of oligosaccharides, and in particular, the invention pertains to the preparation of a high-purity maltose product.
Maltose, an alpha 1-4 linked disaccharide, is a commercially important chemical with numerous industrial and consumer uses, for instance, as a sweetener or in the preparation of maltitol, a different sweetener in confectioneries intended for the low calorie or diabetic market. Maltose is also commercially important in the brewing industry, wherein it is provided as a fermentable component of brewing worts. In these applications, it is desirable to provide maltose in a substantially pure form, i.e., to the substantial exclusion of other carbohydrates. For instance, it is desirable in brewing applications to provide maltose to the substantial exclusion of dextrose. Although dextrose is fermentable, dextrose begins to inhibit yeast growth when present in the brewing wort, for reasons believed to relate to metabolic disturbances of the yeast used in the fermenting process. Similarly, in the preparation of maltitol, it is commercially desirable to provide maltose in a substantially pure form, so that sorbitol and higher molecular weight hydrogenated sugars such as maltotritol are not formed upon hydrogenation of the maltose. For these reasons, the production of maltose in a highly pure form is highly commercially desirable. It is particularly desirable to avoid substantial amounts of glucose, maltotriose, or other lower molecular weight sugars, because of the difficulty in separating maltose from such other carbohydrates.
Generally, maltose is prepared via the enzymatic hydrolysis of starch, whereby certain enzymes, known as beta amylases, convert starch into maltose. Natural starches are composed of two molecules, amylose, a linear saccharide, and amylopectin, a branched starch molecule which for a given starch ordinarily is of higher molecular weight than amylose and in which approximately 4% of the glucoside bonds are alpha 1-6 bonds. With the exception of so-called waxy (corn) or glutinous (rice) specialty starches, most starches found in nature are composed of between 20% and 30% amylose. In the case of amylose, beta-amylase enzymes catalyze hydrolysis from the non-reducing end of the molecule, thus releasing maltose units sequentially until the reducing end is reached. Presumably, if the amylose molecule contains an even number of glucose units, the sole product will be maltose; however, if the amylose molecule contains an odd number of glucose units, then, in addition to maltose, one glucose molecule is released per amylose molecule from the reducing end. In addition to glucose, maltotriose is often seen as an end product due to the relatively slow conversion of maltotriose to glucose and maltose. Moreover, if the amylose were present as a large number of short molecules, such as would be the case if the amylose had been partially hydrolyzed with an acid or alpha-amylase enzyme, then relatively larger amounts of glucose and maltotriose would result upon treatment of the short-chain amylose with beta-amylase. With respect to amylopectin, beta-amylase enzymes release maltose by sequential hydrolysis, but when the sequential hydrolysis reaches an alpha 1-6 branch point, the hydrolysis stops because the enzyme is unable to hydrolyze the branch point. As a result, the highest yield of maltose obtainable from amylopectin is about 50%. The remaining 50% of the original amylopectin exists as large, essentially water-soluble molecules.
For the foregoing reasons, the purity of the maltose product obtained via enzymatic hydrolysis of a starch is related to the number of amylose molecules per given weight of starch, and is limited by the extent of branching in the amylopectin component of the starch. Because of the production of by-products such as glucose and maltotriose, it is difficult to obtain a high-purity maltose from starch without subsequent purification steps. Conventionally, chromatographic processes have been employed to separate maltose from the glucose and other by-products of the beta-amylase hydrolysis product. Chromatographic separations are expensive and difficult to perform, however, thus resulting in an increased cost for a high-purity maltose product.
In recognition of this problem, the prior art has provided a number of attempts to improve the yield of maltose from starch. For instance, yields of maltose from amylopectin can be significantly increased by cleaving (hydrolyzing) the amylopectin structure between branch points with acid or with an alpha-amylase enzyme. This can be done either before treating the starch with a beta-amylase enzyme, or concurrently therewith. By cleaving between branch points, additional non-reducing ends are produced, thus providing more sites for attack by the beta-amylase enzyme. Using this technique, yields of maltose can be increased, but in addition to maltose, small branched molecular products undesirably are produced. Another strategy for increasing yields of maltose for amylopectin is the use of enzymes that hydrolyze the alpha 1-6 branch points. Enzymes such as pullulanse and isoamylase are known; such enzymes are generally known as alpha 1-6 glucosidases.
Such techniques are somewhat effective in improving the yield of maltose. For example, if normal starch (containing about 25% amylose and 75% amylopectin) is liquefied using conventional alpha-amylase or acid liquefaction techniques, and then treated with barley beta-amylase, a yield of about 55% maltose can be expected, the yield depending somewhat on the extent of prior hydrolysis with alpha-amylase. Barley malt-derived beta-amylase enzymes, which contain an alpha-amylase component, will provide a maltose yield of closer to 60%. The use of starch de-branching enzymes will provide even higher yields, ranging up to 75% maltose. Other attempts have focused on the development of new enzymes for use in the production of maltose from starch. One such enzyme (Maltogenase, from Novozymes A/S) is used in combination with an alpha-amylase enzyme, a beta-amylase enzyme, and a pullulanse enzyme to obtain maltose from starch. The maximum yield of maltose using these four enzymes appears to be about 80%.
The use of multiple enzymes on a commercial scale is expensive, both in connection with the purchase of such enzymes and the removal of the enzymes from the maltose product thus prepared. In addition, although the yield of maltose is high relative to other known processes, still the product obtained via the use of such enzymes contains substantial amounts of dextrose and of higher order oligosaccharides (typically having a degree of polymerization (DP) ranging from 3 to about 10). The presence of such other saccharides is undesirable for many purposes, in particular in brewing applications and in maltitol production, and thus expensive chromatographic processes are still required before a high-purity maltose product is obtained. In addition, although it may be feasible to develop new enzymes in an effort to further increase the yield of maltose, such development of new enzymes is extraordinarily expensive, and it is by no means certain that such new enzymes will be more effective than known techniques in preparing maltose products.
For the foregoing reasons, the prior processes for preparation of maltose discussed hereinabove suffer from a number of drawbacks. The present invention seeks to address these drawbacks by providing a process for the production of maltose from starch.
It has now been found that the treatment of starch with an enzyme that consists essentially of a beta-amylase enzyme, and which is to the substantial exclusion or complete exclusion of alpha-amylase enzymes and de-branching enzymes, will yield a product mixture that includes maltose and that is substantially free of glucose and of other malto-oligosaccharides having a DP from 3-10. The maltose may be readily separated from the product mixture thus formed via ultrafiltration to yield a high-purity maltose product in which the maltose content is greater than 70% based upon total carbohydrate and in which the combined content of glucose and oligosaccharides having a DP ranging from 3-10 is below 10%. In some embodiments the maltose product prepared via enzymatic hydrolysis content will have a maltose content greater than 70%. Using the invention, a maltose content greater than 85%, more preferably greater than 90%, and even more preferably greater than 95% may be obtained, and in each case, the combined content of glucose and of oligosaccharides having a DP ranging from 3-10 can be kept below about 10%, preferably below about 5%. The production of high purity maltose in accordance with the invention can be very inexpensive relative to known processes. It has further been discovered that the product formed upon enzymatic hydrolysis of the starch may be spray dried to yield a spray dried maltose product.
In accordance with one embodiment of the invention, a starch is treated with an enzyme that consists essentially of a beta-amylase enzyme, and which is to the substantial exclusion or complete exclusion of alpha-amylase enzymes and de-branching enzymes. The starch is so treated under conditions that are suitable for the hydrolysis of the amylose component of the starch to form maltose, and for the hydrolysis of the amylopectin component of the starch to form maltose and at least one higher molecular weight carbohydrate. Generally, the resulting mixture of starch hydrolysis products will comprise maltose and the higher molecular weight carbohydrates to the substantial exclusion of other malto-oligosaccharides. This mixture may itself have a high maltose content, and may easily be resolved via ultrafiltration into a maltose fraction that has a maltose content greater than 70%, in most cases substantially greater. Moreover, because of the substantial absence of other malto-oligosaccharide by-products in the maltose fraction, such as would be present via conventional processes for maltose production, the mixture may be resolved via ultrafiltration, and no column chromatographic process is required. Upon ultrafiltration, most of the maltose passes through the membrane, leaving some maltose in the retentate. If desired, diafiltration may be used to separate substantially all of the maltose from the high molecular weight carbohydrates in the retentate.
In accordance with anther embodiment of the invention, a starch is treated with an enzyme that consists essentially of a beta-amylase enzyme under conditions that are suitable for the hydrolysis of the amylose and amylopectin components of the starch to form maltose and at least one higher molecular weight carbohydrate. After further optional treatments, such as to remove retrograded amylose, the product mixture that is formed is spray-dried. The spray-dried product readily may be transported and processed.
The invention contemplates the production of maltose from starch. Any suitable starch may be employed in connection with the invention, and thus, for instance, starches such as corn, rice, wheat, tapioca, maize, potato, barley, oat, and, more generally, any starch suitable for enzymatic hydrolysis may be used in connection with the invention. It is not necessary to use a so-called waxy or glutinous starch in connection with the invention, but to the contrary the starch can have any suitable amylose content, such as an amylose content of 10%, 15%, 20%, 25%, or a greater amylose content. It is contemplated that the starch may be a partially derivatized or otherwise modified starch, or may be a starch that has been thinned or enzymatically treated. For instance, a starch that has been lightly oxidized may be employed.
The starch should be liquefied via heat, enzymatic, or acid treatment prior to treatment with the beta-amylase enzyme. Preferably, the starch is liquefied via acid treatment, although low amylose starches may require liquefaction only with heat and may be suitably liquefied at the operating temperature of the enzymatic hydrolysis. Generally, the starch should be liquefied to an extent such that it would remain liquid at the operating temperature of the beta-amylase hydrolysis, but not liquefied to an extent such that the starch is converted to saccharides having so low a degree of polymerization that it is difficult to separate such saccharides from maltose via ultrafiltration. In other words, the degree of liquefaction should be such that, upon enzymatic hydrolysis with the beta-amylase enzyme, the combined content of glucose and oligosaccharides in the DP 3-10 range does not exceed about 10%, and preferably does not exceed about 5%. It has been found acceptable to liquefy the starch to a dextrose equivalent (DE) value of about 2 as measured via conventional techniques. Generally, the DE of the starch should be kept below about 1, and should thus range between 0 and about 1, although it may be difficult to measure the DE with precision in this range. For corn starch, it is preferable that the starch is liquefied in an aqueous solution at a liquefaction temperature ranging from about 220xc2x0 F. to about 320xc2x0 F., and for a time ranging from about 5 minutes to about 30 minutes.
The starch solids level preferably ranges initially from about 5% to about 30%, more preferably, from about 15% to about 30%. It is believed that a lower solids level in the starch slurry will result in a higher purity maltose upon enzymatic hydrolysis. While it is not intended to limit the invention to a particular theory of operation, it is believed that a lower solids content requires a lesser degree of liquefaction to attain the desired viscosity range. In the case of dent corn starch, it has been found that a viscosity window of between 25 and 45 centipoise (Norcross Shell Cup) is optimal. In the case of waxy starches, viscosities outside this range may be acceptable. The pH of the starch slurry should be adjusted to a level sufficient to provide controlled acid hydrolysis of the starch in the presence or absence of catalyzing alpha amylase enzymes; most preferably, under a given set of conditions, the variability of the slurry pH should be no more than +/xe2x88x920.1 pH, with the precise pH value depending upon the starch source, the slurry solids, and the operational conditions of the liquefaction equipment employed. As a practical matter, the pH can vary more widely while still resulting in a satisfactory maltose product. Preferably, the starch liquefaction is monitored via viscosity and adjusted accordingly.
In accordance with one embodiment of the invention, the starch is liquefied with an alpha-amylase enzyme to reduce the molecular weight of the starch, thereby reducing the viscosity of the starch and thereby permitting processing at a higher solids level. Suitable commercial liquefying enzymes may be obtained from Genencor International or from Novozymes A/S. Any suitable liquefaction conditions may be employed. The dosing level of the alpha-amylase enzyme depends upon the desired solids level and the desired maltose purity, and desirably ranges from about 0.005% to about 0.02% of a commercial strength enzyme by dry solids basis starch. In this embodiment, the alpha-amylase enzyme preferably is quenched prior to saccharification via any suitable quenching procedure. For instance, when the starch is liquefied at a temperature less then 250xc2x0 F. and 5 minutes residence, the alpha-amylase enzyme is quenched by reducing the liqefact pH to less than 4.0 and holding at a temperature of from 180 to 190xc2x0 F. for at least about 15 minutes.
Upon liquefaction, the liquefact is immediately cooled and the pH is adjusted to the optimum conditions for beta-amylase activity. The starch then is treated with the enzyme under any conditions suitable to result in the hydrolysis of this liquefied starch to form maltose. A preferred enzyme is OPTIMALT BBA, available from Genencor International, Inc. The enzyme may be added in any amount sufficient to achieve this result, but generally, the dosing of the enzyme should be in excess of the minimum viscosity limited conversion of approximately two Genencor International, Inc. OPTIMALT BBA Diastatic Power units per kilogram of starch, the Diastatic Power units being defined as being the amount of enzyme contained in 0.1 ml of a 5% solution of the sample enzyme preparation that will provide sufficient reducing power to reduce 5 ml of Fehling""s solution when the sample is incubated with 100 ml of substrate for one hour at 20xc2x0 C.
The enzymes should be allowed to act on the starch for any amount of time suitable to form maltose. Under the preferred reaction conditions discussed hereinabove, saccharification generally is 90% complete within 4 hours. The optimum temperature and pH of the starch hydrolysis will vary depending on the particular beta-amylase enzyme employed, but typically the temperature will range from about 55xc2x0 C. to about 65xc2x0 C. and the pH will range from about 5.0 to about 6.0.
At the conclusion of the enzymatic hydrolysis, a product that contains maltose and at least one higher molecular weight carbohydrate will be provided. Optionally, but preferably, this product mixture is clarified and decolored by any suitable procedure, such as carbon treatment, filtration, centrifugation, and/or precipitation, before it is further processed. Maltose content in the product may be at least about 30% by weight, and in some cases much higher. If the enzyme is allowed to act under optimum conditions for an optimum reaction time, the maltose content of such product may be equal to or greater than about 50%, in some cases equal to or greater than 60%, in some cases equal to or greater than 65%, in some cases greater than or equal to about 70%, and even in some cases equal to or greater than about 75%, while the content of higher molecular weight carbohydrates having a dextrose equivalent value (DE) less than 5 may be greater than about 20%. The combined content of glucose and of oligosaccharides in the DP 3-10 range is below about 10%, and preferably is below 5%.
It is contemplated that in some instances a lower maltose content in the product may be desired. In such case, the enzyme may be quenched prior to the optimum reaction time, such as by adjusting the pH or applying heat. In a highly preferred embodiment, a correlation between enzyme reaction time and resulting maltose content is prepared or is provided in advance. Such a correlation between reaction time and maltose content may be determined empirically, for instance, by performing at least two experiments in which the enzyme is quenched at different reaction times each less than the optimum reaction time, and measuring the maltose content in each case. Once the correlation has been prepared or provided in advance, a desired maltose content may be obtained by quenching the enzyme after a reaction time determined with reference to the predetermined correlation. It is also contemplated that the maltose content may be controlled by conducting the liquefaction under less than optimal conditions, although such is not preferred inasmuch as a higher content of glucose and/or lower molecular weight sugars in the product mixture may thereby result. In accordance with this embodiment of the invention, various products having differing maltose contents can be prepared. For instance, it is contemplated that a product in which the maltose content is 10%, 20%, or 30% may be prepared. In each case the combined content of glucose and of oligosaccharides in the DP 3-10 range can be kept below about 10%, and preferably below 5%.
Retrograded amylose may be found as a by-product of the enzymatic hydrolysis. In accordance with one embodiment of the invention, at least some of the retrograded amylose is separated from the product mixture. For instance, the saccharified solution may be maintained at a temperature below about 140xc2x0 F. to allow at least a portion of the retrograded amylose to crystallize. The crystallized amylose then may be separated from the saccharified starch mixture by any suitable technique, such as via microfiltration, by which is contemplated separation at a resolution sufficient to separate the retrograded amylose but not sufficient to separate maltose from higher molecular carbohydrates in the product mixture. Alternatively, the retrograded amylose may be separated via centrifugation, using any technique known in the art or otherwise found to be suitable.
In accordance with one embodiment of the invention, a high purity maltose product is separated from the product mixture. Most preferably, a maltose product is separated from the product mixture via ultrafiltration of the product mixture, by which is contemplated separation of the maltose from higher molecular weight carbohydrates using a membrane or other suitable separation medium that is effective for this purpose. Generally, a membrane having a molecular weight cut off (MWCO) of 10,000 or less, preferably a MWCO of 5000 or less, is suitable. Suitable commercially available membranes available from Sydnar Filtration and from Osmonics De Sal. Upon ultrafiltration, the permeate includes a high purity maltose liquid, which may be evaporated to a high solids content (greater than 55%) that crystallizes upon cooling. The purity of the maltose obtained upon crystallization can be as high as 98%, with dextrose levels below the detection level of conventional HPLC analysis. The retentate typically will include a higher molecular weight carbohydrate having a DE less than 5 and some retained maltose. If desired, the retentate may be diafiltered to recover additional maltose by flushing the filter with excess water.
In accordance with another embodiment of the invention, the product mixture obtained upon enzymatic hydrolysis of the starch is spray-dried. Surprisingly, it has been found that it is possible to spray dry a maltose product prepared as discussed hereinabove. While it is not intended to limit the invention to a particular theory of operation, it is believed that the composition of the product is especially suitable for spray drying, perhaps because of the relatively low amount of maltotriose, which is substantially more hygroscopic than maltose. The high molecular weight carbohydrate in the product may function as an effective spray drying enhancer. Prior to spray drying, the product mixture may be clarified and decolored, and retrograded amylose may be removed as discussed above.
Carbohydrate percentages given herein are expressed on a dry solids basis per total carbohydrate weight.
The following examples are provided to illustrate the invention, but should not be construed as limiting in scope.